Novel Nitric Oxide Donors of Phenylsulfonylfuroxan and 3-Benzyl Coumarin Derivatives as Potent Antitumor Agents

Article abstract of DOI:10.1021/acsmedchemlett.8b00125

In this work, five new hybrids of phenylsulfonylfuroxan merging 3-benzyl coumarin and their seco-B-ring derivatives 2-6 were designed and synthesized. Among them, compound 3 showed the most potent antiproliferation activities with IC₅₀ values range from 0.5 to 143 nM against nine drug-sensitive and four drug-resistant cancer cell lines. Preliminary pharmacologic studies showed that these compounds displayed lower toxicities than that of lead compound 1. Compound 3 obviously induced the early apoptosis and hardly affected the cell cycle of A2780, which was significantly different from compound 1. Especially, it gave 559- and 294-fold selectivity antiproliferation activity in P-gp overexpressed drug-resistant cancer cell lines MCF-7/ADR and KB-V compared to their drug-sensitive ones MCF-7 and KB, implying that compounds 2-6 might have an extra mechanism of anti-MDR-cancer with P-gp overexpression.

Full text of DOI:10.1021/acsmedchemlett.8b00125

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Novel nitric oxide donors of phenylsulfonylfuroxan and 3-
benzyl coumarin derivatives as potent antitumor agents
Yalan Guo, Yujie Wang, Haihong Li, Ke Wang, Qi Wan, Jia Li, Yubo Zhou, and Ying Chen
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00125 • Publication Date (Web): 20 Apr 2018
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ACS Medicinal Chemistry Letters
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Novel nitric oxide donors of phenylsulfonylfuroxan and 3-benzyl
coumarin derivatives as potent antitumor agents
Yalan Guo,† Yujie Wang,‡ Haihong Li,† Ke Wang,† Qi Wan,† Jia Li,‡ Yubo Zhou,‡* Ying Chen†*
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†Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203, China
‡Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences, Shanghai 201203, China
KEYWORDS: Phenylsulfonylfuroxan, 3-benzyl coumarin, Anti-cancer, Multi-drug resistance, P-gp overexpression
ABSTRACT: In this work, five new hybrids of phenylsulfonylfuroxan merging 3-benzyl coumarin and their seco-B-ring
derivatives 2-6 were designed and synthesized. Among them, compound 3 showed the most potent antiproliferation ac-
tivities with IC50 values range from 0.5 nM to 143 nM against nine drug-sensitive and four drug-resistant cancer cell lines.
Preliminary pharmacologic studies showed that these compounds displayed lower toxicities than that of lead compound 1.
Compound 3 obviously induced the early apoptosis and hardly affected the cell cycle of A2780, which was significantly
different from compound 1. Especially, it gave 559 and 294 folds selectivity antiproliferation activity in P-gp overexpressed
drug-resistant cancer cell lines MCF-7/ADR and KB-V compared to their drug-sensitive ones MCF-7 and KB, implying
that compounds 2-6 might have an extra mechanism of anti-MDR-cancer with P-gp overexpression.
Nitric oxide (NO), which was reported by Furchgott
and Zawadzki as an endothelium-derived relaxing factor
(EDRF) in 1980,1-2 plays important roles in diverse physio-
logical and pathophysiological processes.3-8 Additionally,
NO can down-regulate PI3K/Akt pathway and up-regulate
MEK/ERK pathway.9-11 Hideo Baba et al. reported that the
combined administration of NO donor and MEK inhibitor
can synergistically inhibit the viability of cancer cells
through downregulating both PI3K/ Akt and MEK/ERK
pathways.12 (3,4-Bis(phenylsulfonyl)-1,2,5-oxadiazole 2-
oxide (phenylsulfonylfuroxan) as an important NO donor,
was widely used in the design of anticancer agents.13-17 We
previously reported that phenylsulfonylfuroxan and cou-
marin hybrid 1 showed remarkably antitumor activity
through multi-target mechanism containing disruption of
MEK pathway. However, its MEK inhibitory activity was
Scheme 1). The structure optimization aimed at develop-
ing stronger synergistic antiproliferation activity with
both NO donor and MEK inhibitory activitiy compared to
lead compound 1. Besides, concerning coumarin core in-
tegrity for sustaining anti-cancer activity, two seco-B-ring
derivatives were also synthesized.
As showed in Scheme 1, compounds 8a-c, synthesized
according to a previously described procedure,22-23 were
treated with 2-chloro-1-ethanol to provide intermediates
9a-c. Compound 9c was reduced by stannous chloride
dehydrate to form 9d and sulfonylated with N-methyl-2-
oxooxazolidine-3-sulfonamide24 to obtain 9e. Compound
11 was prepared from resorcinol via Friedel-Crafts reaction,
4-hydroxyl protection and 2-hydroxyl methylation. After
aldol condensation of 11 with benzaldehyde or 4-
fluorobenzaldehyde, deprotection of the 4-hydroxyl gave
12a, b. Seco-B-ring compounds 13a, b were synthesized by
the same procedure used to obtain 9a-c. Finally, 9a-b, e
and 13a, b were merged with phenylsulfonylfuroxan to
provide compounds 2-6. Meanwhile, compound 15 con-
taining phenylsulfonylfuroxan-linker fragment was also
relatively weak.18
Considering 4-fluorobenzyl at 3-
position of coumarin skeleton in G8935, which was a MEK
inhibitor19-20, occupied a new binding pocket in the MEK
docking model,21 we also introduced several benzyl
groups covering 4-fluorobenzyl to the same position of
lead compound 1 and obtained five new derivatives (2-6,
1
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Page 2 of 6
synthesized as a reference for bio-evaluation analyses.
inhibit the proliferation of drug-resistant cancer with P-
gp overexpression.26-31 This promoted us to detect P-gp
expression of the drug-resistant and their drug-sensitive
cancer cell lines mentioned above. As expected (see Fig-
1
Structures of 2-6 were confirmed by H NMR, 13C NMR
1
2
3
1
and MS spectra. In H NMR, the chemical shift of two
alkenyl hydrogens of α,β-unsaturated ketone in com-
4
5
6
7
8
9
pound 6 were 7.65 and 7.43 ppm, respectively, and the
ure S4 , P-gp expression was outstanding in KB-V and
)
coupling constantis 15.8 Hz, indicating
a
trans-
MCF-7/ADR, to which compounds 2-6 showed significant
selective antiproliferation potency compared to KB and
MCF-7; while P-gp overexpression wasn’t observed in
A2780/CDDP, MDA-MB-231/GEM, A2780 and MDA-MB-
231, which having no relevant selective antiproliferation
activities for 2-6. Further work is under way to find the
possible pharmacologic mechanism of these compounds
against P-gp overexpression MDR cancer.
conformation of the ethylenic double bond structure.
Then 2-6 were screened for their bioactivities against
nine drug-sensitive solid cancer cell lines, two hematolog-
ical tumors, four drug-resistant cancer and four nontu-
morigenesis cell lines with lead compound 1 and NO do-
nor 15 as references, and cisplatin, doxorubicin, gemcita-
bine, vincristine, SAHA, lenalidomide and CC-88525 were
chosen as positive controls. Except for MCF-7 and KB
(Table 1), compounds 2-6 all showed higher antiprolifera-
tion activities (0.5 to 35.8 nM) than that of 1 and 15. Par-
ticularly, compound 3 with 4-fluorobenzyl at 3-position of
coumarin core was the most potent compound (0.8 to 8.3
nM). In A549, OVCA429 and MDA-MB-231, 2-6 all dis-
played significant activities in single digit nanomolar lev-
els of IC50 values. While in HeLa, SKOV3, OVCA433 and
A2780, 2 and 3 bearing 3-benzyl coumarin skeleton
showed slightly better activities than that of their seco-B-
ring derivatives (5 and 6). Introduction N-
methylsulfomicamino group into the 3-position of phenyl
group (4) resulted in a slightly decreasing activities rela-
tive to compounds 3 and 6 bearing 4-fluorophenyl group.
Additionally, they had strong bioactivities with a range
from 5.1 to 156.8 nM of IC50 values against two hematolog-
ical tumor cell lines MV-4-11, MM-1S and four drug-
resistant cancer cell lines A2780/CDDP, MDA-MB-
231/Gem, MCF-7/ADR and KB-V (Table 2, 3). Notably, the
selective ratios of IC50 values about 3 were 559 and 294
(Table 3, 2.9 and 3.2 μM in MCF-7 and KB vs 5.1 and 11.0
nM in MCF-7/ADR and KB-V).Whereas, compounds 2-6
were almost at the same activity levels against
A2780/CDDP vs A2780 and MDA-MB-231/Gem vs MDA-
MB-231. The distinct selectivities presumed that new NO
donors 2-6 probably had an extra pathway to inhibit cer-
tain MDR cancer. Moreover, 2-6 expressed far lower toxic-
ities than compound 1 in HUVEC, T29, WI-38 and MCF-
10A (Table 4, 0.19 to 20µM), which indicated they have a
noteworthy selectivity between tumor and nontumor-
igenesis cell lines.
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Scheme 1. Design (a) and synthesis (b) of 3-benzyl
coumarin and phenylsulfonylfuroxan hybrids and
their seco-B-ring derivativesa.
O
3
A
B
C
R1
R2
N
O
O
O
F
A
B 3
O
C
G8935
O
N
combination
O
O
O
3-phenyl Coumarin - Phenylsulfonylfuroxan
Hybrids
N
O
SO2Ph
seco-B-ring
O
O
N
O
O
O
O
R1
N
O
1
SO2Ph
O
N
O
O
R2
O
N
O
seco-B-ring
derivatives
SO2Ph
(a)
Br
R1
R1
R2
a, b
c
R2
HO
R1
HO
O
O
R2
O
O
O
7a R1=H, R2=H
7b R1=H, R2=F
7c R1=NO2, R2=H
8a R1=H, R2=H
8b R1=H, R2=F
8c R1=NO2, R2=H
9a R1=H, R2=H
9b R1=H, R2=F
9c R1=NO2, R2=H
d
e
9e R1=NHSO2NHCH3
,
9d R1=NH2, R2=H
R2=H
O
O
O
O
f, g, h
i, j
c
HO
OH
HO
R
O
O
O
HO
O
O
R
13a R=H
13b R=F
10
11
12a
12b
R=H
R=F
R1
R2
O
N
O
O
O
O
2 R1=H, R2=H
N
SO2Ph
3
R1=H, R2=F
O
O
PhO2
O
S
PhO2
O
S
SO2Ph
O
4 R1=NHSO2NHCH3, R2=H
k
l
N
N
N
N
O
O
O
14
15
O
N
O
O
R
O
N
O
5 R=H
6 R=F
SO2Ph
(b)
aReagents and conditions: (a) ethyl 3-oxobutanoate (1.0
equiv), NaH (1.2 equiv), dry THF, 60 °C, 2 h; (b) resorcinol
(1.0 equiv), 70% H2SO4, rt, 2 h, 35% ~ 95% for two steps (a
and b); (c) 2-chloro-1-ethanol (1.0 equiv), K2CO3 (3.0 equiv),
KI (0.1 equiv), DMF, reflux, 2 h ~ 10 h, 76% ~ 100%; (d) stan-
nous chloride dehydrate (4.0 equiv), DMF, rt, 6 h, 99%; (e)
N-methyl-2-oxooxazolidine-3-sulfonamide (2.0 equiv), NEt3
(3.0 equiv), MeCN, 80 °C, 8 h, 99%; (f) ZnCl2 (1.5 equiv),
HOAc, reflux, 70 min, 51%; (g) chloromethyl methyl ether
(2.0 equiv), K2CO3 (2.5 equiv), actone, rt, overnight, 84%; (h)
CH3I (1.2 equiv), K2CO3 (3.0 equiv), DMF, 80 °C, 30 min, 82%;
(i) benzaldehyde or 4-fluorobenzaldehyde (1.05 equiv), 60%
KOH aqueous solution (2 mL/mM compound 11), EtOH, 2 ~
5 h, rt; (j) conc. HCl : EtOH = 1:25 (v/v), reflux, 30 min, 80% ~
85% for two steps (i and j); (k) 14 (1.3 equiv), DBU (1,8-
diazabicyclo[5.4.0]undec-7-ene) (2.0 equiv), anhydrous DCM,
rt, 3.5 h ~ 12 h, 51% ~ 85%; (l) 2-methoxyethan-1-ol (1.1 equiv),
DBU (2.0 equiv); anhydrous DCM, rt, overnight, 90%.
Assay of NO release showed that 2-6 produced much
less NO compared to control 1 (See Figure S1), and the
antiproliferation activities of compound 3 was declined
with the increasing concentration of scavenger c-PTIO in
both A2780 and A2780/CDDP (Figure 1). However, com-
pounds 2-6 showed much weaker MEK inhibiting potency
than that of 1, which elucidated activities of 2-6 might be
mainly related to the release of NO. Furthermore, com-
pounds 3 and 6 could remarkably induce cell apoptosis,
but hardly affect cell cycle. Interestingly, 3 and 6 seemed
to mainly induce early apoptosis, while reference 1 mainly
late apoptosis (see Figure S3
)
. Recently, some com-
pounds including Dp44mT were reported to specifically
2
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ACS Medicinal Chemistry Letters
In conclusion, novel NO donors 2-6 not only displayed
study results were different from lead compound 1. Espe-
cially, compound 3 exhibited obvious selectivity ratios of
anti-cancer potency in drug-resistant and their drug-
sensitive cell lines MCF-7/ADR vs MCF-7 and KB-V vs KB
with 559 and 294 folds, respectively. Western Blot analy-
sis discovered the overexpression of P-gp in MCF-7/ADR
and KB-V, while did not overexpress in MCF-7 and KB.
The resu-
1
2
3
4
5
6
7
8
significant antiproliferation activities in nine drug-
sensitive tumor cell lines, but also showed strong activi-
ties against four drug-resistant tumor cell lines with na-
nomolar even sub-nanomolar level IC50 values. The less
NO-releasing levels and lower toxicity on nontumorigen-
esis cell lines compared to lead compound 1 suggested
that they had a better selectivity against malignant cells in
vitro. Notably, some of their preliminary pharmacologic
Table 1. Antiproliferation activities for 2-6 against nine solid cancer cell linesa
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
IC50 (nM)
Compd.
MDA-
MB-231
HeLa
SKOV3
A549
OVCA429
OVCA433
A2780
MCF-7
KB
15
8826
62.2
22.9
2.8
3909
47.3
17.3
8.3
4778
45.8
2.0
5173
41.1
1.7
4389
130.8
1.1
1635
20.9
12.7
6.6
1233
85.9
6.0
0.8
1.1
3503
445
2351
2853
3131
1865
1413
NA
3691
127
1
2
4542
3234
1143
2182
1665
NA
3
3.7
3.9
3.3
4
15.4
17.8
6.9
19.2
35.8
25.0
1440
NA
7.9
6.1
16.3
10.1
10.2
8.7
5
3.3
4.4
4.0
7296
NA
NA
NA
0.5
6
3.5
6.6
14.5
2549
NA
1.4
Cisplatin
Doxorubicin
Gemcitabine
2741
NA
13220
NA
NA
NA
24630
NA
NA
NA
36.4
NA
879
NA
NA
NA
NA
NA
NA
NA
Vincristine
a
NA
NA
NA
NA
NA
0.65
MTT assay. The data are the mean of triplicate determinations. NA: not available. HeLa: human cervical cancer cell lines;
SKOV3, OVCA429, OVCA433 and A2780: human ovarian cancer cell lines; A549: human non-small cell lung cancer cell lines;
MDA-MB-231 and MCF-7: human breast cancer cell lines; KB: human oral epidermoid cancer cell lines.
Table 2. Antiproliferation activities for 2-6 in hematological tumor cell linesa
IC50 (nM)
MV-4-11
1
2
3
4
5
6
SAHA
NA
Lenalidomide
> 20000
CC-885
0.3
28.3
24.5
27.2
66.3
29.3
29.6
MM-1S
21.0
12.0
143.0
24.0
28.0
25.0
2844
NA
NA
a MTS assay. The data are the mean of triplicate determinations. NA: not available. MV-4-11: human myeloid leukemia cell lines;
MM-1S: human myeloma cell lines.
Table 3. Antiproliferation activities for 2-6 against drug-resistant tumor cells a
IC50 (nM)
Selectivity ratio
IC50(MDA-MB-231)
/
Compd.
A2780/
CDDP
MDA-MB-
231/Gem
MCF-
7/ADR
IC50(A2780)
IC50(A2780/CDDP)
/
IC50(MCF-7)
/
IC50(KB)/
KB-V
IC50(MDA-MB-
IC50(MCF-7/ADR) IC50(KB-V)
231/Gem)
15
1
4820
85.7
94.0
22.8
47.2
51.7
4747
85.9
25.2
6.9
7845
156.8
16.3
5.1
1959
24.9
16.0
11.0
0.3
0.2
0.1
0.3
0.2
0.2
0.4
0.3
1.0
0.2
0.1
0.02
0.05
0.02
0.5
2.8
1.9
5.1
2
3
144.2
559.4
62.5
103.6
29.5
283.9
294.0
17.5
157.0
164.9
4
5
50.4
10.5
93.0
50.1
18.0
47.9
65.4
13.9
10.1
6
34.1
201370
(r.r. = 79)
Cisplatin
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
>100000
(r.r. > 113)
Doxorubicin
Gemcitabine
Vincristine
NA
NA
NA
>50000
(r.r. > 1373)
NA
NA
417.0(r.r.
= 646)
NA
a
MTT assay. The data are the mean of triplicate determinations. r.r. : resistant ratio, r.r. = IC50 (drug-resistant cell lines) / IC50 (drug-sensitive
cell lines), NA: not available. A2780/CDDP: cisplatin resistant human ovarian cancer cell lines; MDA-MB-231/Gem: gemcitabine
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resistant human breast cancer cell lines; MCF-7/ADR: doxorubicin resistant human breast cancer cell lines; KB-V: vincristine
resistant human oral epidermoid cancer cell lines
1
2
3
Table 4. Antiproliferation activities for 2-6 in nontumorigenesis cells
4
5
6
7
8
9
Le-
nalimide
1
2
3
4
5
6
cisplatin
CC-885
IC50 (µM)
HUVECa
T29a
WI-38b
MCF-10Ab
0.13
1.8
0.44
> 5
0.27
> 5
0.19
> 5
0.41
2.5
0.36
3.5
1.10
1.8
NA
NA
NA
NA
0.09
> 20
> 20
1.36
0.44
0.56
NA
> 20
> 20
0.66
> 20
> 20
NA
NA
NA
NA
> 20
0.005
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a MTT assay; b MTS assay. The data are the mean of triplicate determinations. NA: not available. HUVEC: human umbilical vein
endothelial cell lines; T29: human immortalized but non-tumorigenic ovarian epithelial cell lines; WI-38: human lung fibro-
blasts; MCF-10A: human non-tumorigenic breast epithelial cell lines
P-gp, P-glycoprotein; MDR, multi-drug resistant; PI3K, phos-
phatidylinositol-3-kinase; Akt, protein kinase B; MEK, Mito-
gen-activated protein kinase kinase; ERK, extracellular regu-
lated protein kinases; NMR, nuclear magnetic resonance;
HSQC, heteronuclear single quantum correlation; THF, tet-
rahydrofuran; DMF, N,N-dimethylformamide; DCM, di-
chloromethane; SAHA, suberoylanilide hydroxamic acid;
DMSO, dimethyl sulfoxide; PI, propidium iodide; HRMS,
high resolution mass spectra
Figure 1. Antiproliferation activities of compound 3 at the
concentration of 50 nM together with different concentra-
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
anti-proliferative assay, inhibition activities of colony, nitrite
measurement, cell apoptosis and western-blot analysis,
HRMS, 1H &13C NMR and HSQC spectra of compounds (PDF).
AUTHOR INFORMATION
Corresponding Author
Email: yingchen71@fudan.edu.cn
Author Contributions
Y. L. Guo† and Y. J. Wang‡ contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
Funding from "Major New Drugs Innovation and Develop-
ment" of National Science and Technology Major Projects
(2018ZX09711002-007-005); The authors wish to thank Zhi-
min Shao and Gong Yang for providing cancer cell lines.
11. Lim, K. H.; Ancrile, B. B.; Kashatus, D. F.; Counter, C. M.
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Novel nitric oxide donors of phenylsulfonylfuroxan and 3-benzyl
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Yalan Guo,† Yujie Wang,‡ Haihong Li,† Ke Wang,† Qi Wan,† Jia Li,‡ Yubo Zhou,‡* Ying Chen†*
†Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203, China
‡Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences, Shanghai 201203, China
KEYWORDS: Phenylsulfonylfuroxan, 3-benzyl coumarin, Anti-cancer, Multi-drug resistance, P-gp overexpression
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